pRcCMV myc-Plk1 wt (Nigg RG6)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||41160||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 5527
- Total vector size (bp) 7746
Vector typeMammalian Expression
Selectable markersNeomycin (select with G418)
Growth in Bacteria
Alt namepolo-like kinase 1
Alt nameserine/threonine-protein kinase 13
SpeciesH. sapiens (human)
Insert Size (bp)2219
GenBank IDNM_005030.3 X73458.1
- Promoter CMV
/ Fusion Protein
- Myc (N terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site HindIII (not destroyed)
- 3′ cloning site XbaI (unknown if destroyed)
- 5′ sequencing primer CMV-F
- 3′ sequencing primer BGH-rev (Common Sequencing Primers)
cDNA fragments spanning parts of the catalytic domains of human protein kinases were amplified by PCR, as described by Schultz and Nigg (1993). A 144 bp fragment (named HsPK28) showing 88% nucleotide identity to Drosophila polo was used to probe a λgt 10 library prepared from a human nasopharyngeal carcinoma (Hitt et al., 1989). Approximately 500,000 plaques were screened by plaque hybridization (Sambrook et al., 1989), and 17 phage showing strong hybridization were purified. DNA was prepared using Lambdasorb (Promega Biotech, Madison, WI).
One plasmid (referred to as Plk1-pGEM) with a 2143 bp insert contained the entire coding sequence for a protein kinase closely related to Drosophila polo. This insert was sequenced on both strands by the method of Chen and Seeburg (1985), using the Sequenase kit (United States Biochemicals).
To express a full-length Plk1 protein, the 1998 bp fragment of Plk1-pGEM was introduced into the pRc/CMV vector (Invitrogen Corporation, San Diego), to create Plk1-CMV. An N-terminally myc-tagged Plk1-CMV was also constructed. In the resulting mycplk1 protein, the N terminus of Plk1 is extended by the oligopeptide MEQKLISEEDLNMNSCSPGS (Evan et al., 1985).
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pRcCMV myc-Plk1 wt (Nigg RG6) was a gift from Erich Nigg (Addgene plasmid # 41160 ; http://n2t.net/addgene:41160 ; RRID:Addgene_41160)
For your References section:Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5. Golsteyn RM, Schultz SJ, Bartek J, Ziemiecki A, Ried T, Nigg EA. J Cell Sci. 1994 Jun;107 ( Pt 6):1509-17. PubMed 7962193